Studies by us and others suggest that bacterial flora (microbiota) influences HIV expression in the female genital tract. Our studies show that in HIV-infected women, the amount of HIV in lower genital tract secretions is positively associated with the amount of Mycoplasma hominis, negatively associated with actobacilli, but not associated with Gardnere//a vaginalis. While intriguing, the previous studies only assessed the association of HIV with three of the bacteria types that can be present in genital tract. More than 20 different bacterial species can make up the genital flora in women. Importantly, the combinations of these bacteria that are found in the genital tract are variable. Also, several of the major types of bacteria can not be cultured, or are difficult to culture, and therefore have not been studied in relation to HIV. In aim 1, we will use molecular techniques (real time PCR and pyrosequencing) to identify genital tract bacteria, thus avoiding culture, in order to investigate the relationship between levels of genital tract HIV and the microbiota. In aim 2 we will investigate the hypothesis that the effects of bacterial flora on HIV expression in the female genital tract are not direct, but are associated with and potentially caused by changes in specific mmune molecules; recent studies by us indicate that HIV levels in genital secretions are associated with certain immune mediators. Multiplex luminex assays will be used to assess cytokines, chemokines and nnate immune mediators in genital tract secretions, which will be compared with HIV and bacterial flora data. We also will collaborate with Dr. Hope to detemine the impact of sialidases on the protective activity of mucous. In aim 3, in collaboration with Dr. Lurain of Core C, we will assess the effects of genital secretions on HIV replication in both ex vivo cervical explants and in U1 cells, and determine the identity of the stimulatory activities. Lastly, in aim 4, in associationwith Drs. Novak and Baum of Project III, we will investigate the relationship of genital tract flora with anti-HIV immunity in highly exposed HIV-seronegative women. This project will lead to a better understanding of the factors that affect heterosexual transmission of HIV. This is important since understanding the impact of different bacterial species will allow us to understand how we could manipulate that microbial environment to reduce the risk of transmission or increase the efficacy of vaccines or microbicides. RELEVANCE (See instructions): Each year, millions of people are infected with HIV by sexual transmission. This work will lead to a better understanding of the factors that affect sexual transmission of HIV and could lead to treatments that reduce HIV transmission.